Present bioluminescent assay methods for determining the presence, number or biomass of microbial cells in a sample which may also contain non-microbial cells require longer processing times than desired. These methods are generally based on the detection of microbial nucleotides, such as adenosine triphosphate (ATP). In a typical assay, microbial cells can be treated so as to release their ATP into solution where it can react with bioluminescent assay reagents, such as firefly luciferase enzyme and luciferin substrate, in the presence of magnesium and oxygen, to produce photons. These photons are detectable and their numbers can be correlated with the amount of ATP, and in turn, with the number or mass of cells originally present. See generally, U.S. Pat. Nos. 3,745,090, 3,971,703, 4,014,745, 4,264,727, 4,303,752 and 4,501,813, and Leach, J. Appl. Biochem. 3, 473-517 (1981).
The longer-than-desired processing times occur because of measures that must first be taken to "remove" from the assay nucleotides of an non-microbial cells which are present in the sample. U.S. Pat. No. 3,745,090 suggests removal of non-microbial ATP by: treating the sample to release ATP specifically from non-microbial cells; hydrolyzing or "inactivating" the released ATP by addition of an "ATP-hydrolyzing enzyme" such as an ATPase or, more commonly, an apyrase, both of which are hydrolase enzymes; and then inactivating or destroying the enzyme so that it does not affect the ATP later released from the microbial cells. The microbial ATP would then be released and detected by means of a bioluminescent assay.
U.S. Pat. No. 3,745,090 suggests certain approaches for inactivating or destroying apyrase activity. Some of these suggested approaches would act by nonspecifically and irreversibly destroying all proteins in the sample (including the enzymes), such as by heat or acid treatment. Such approaches are inconvenient, often hazardous, and radical in that they would very likely destroy microbial cells as well, thereby affecting the sensitivity and accuracy of the assay.
While this patent also suggests the use of "enzyme inhibitors", apparently none have yet been shown to be inhibitory to an ATP-hydrolyzing enzyme, e.g., apyrase, yet suitable for use in such methods, e.g., in terms of being compatible with the reactants, reactions and/or readings of a subsequent bioluminescent assay.
As a result, in spite of the approaches suggested in U.S. Pat. No. 3,745,090, the typical approach in commercial applications to negate the potential effect of the commonly used hydrolase enzyme apyrase on microbial ATP has been to simply employ low concentrations of apyrase, as set forth in U.S. Pat. No. 4,303,752. At these low concentrations the time needed for apyrase to inactivate the non-microbial ATP becomes correspondingly longer. When the inactivation of non-microbial ATP is complete however, microbial ATP can then be released and quickly assayed in spite of the fact that a portion of it will be inactivated in the process by the still-present, still-active apyrase.
The optimal amount of apyrase that is used in a particular application is therefore generally the amount that allows the shortest possible "inactivation time" for non-microbial nucleotides, while still allowing microbial ATP to be detected. As a result, apyrase concentrations are generally quite low, and the inactivation of non-microbial ATP in turn becomes the major time-consuming step in the overall procedure Indeed, many of the commercially available bioluminescent assays for the detection of microbes, e.g., in body fluids and food samples, employ time periods on the order of 10-60 minutes for the complete inactivation of non-microbial ATP by apyrase. This can be compared to the other steps of a typical assay, such as the adding and mixing of reagents, the release of microbial ATP and the production and counting of photons, which are generally accomplished in a matter of minutes, if not seconds, using automated procedures specifically adapted to these assays. In many applications, e.g., where large numbers of samples have to be routinely processed, it would be greatly beneficial to be able to achieve shorter "inactivation times" for non-microbial nucleotides, preferably in a manner compatible with automated assay procedures.
Clearly a method is desirable for the rapid inactivation of non-microbial nucleotides, where the method used for the inactivation can itself be specifically, quickly and effectively negated in a manner that does not interfere with a subsequent assay for microbial nucleotides.